Meningitis is an infection of the meninges, the thin lining that surrounds the brain and spinal cord. Several kinds of bacteria can cause meningitis, and N. meningitidis is one of the most important. Others are Streptococcus pneumoniae and Haemophilus influenzae type b. There are several subgroups of N. meningitidis, which are differentiated by the structure of the capsular polysaccharide that surrounds the bacterium.
Meningitis is caused by both viruses and bacteria. The two major types of bacteria causing meningitis are Haemophilus influenzae and Neisseria meningitidis. In the case of H. influenzae only one serotype, type b, is important, whereas with N. meningitidis twelve serogroups have been identified, of which groups A, B, C, and W135 are known to cause epidemics. The various serotypes have different geographical prevalence, e.g., type B and C are dominant in Europe and North America and Type A in Africa and South America. The serotyping is based on the structure and antigenicity of the capsular polysaccharide (CPS) surrounding the bacteria, and the CPS can also be used as a vaccine against the bacteria. Especially efficient vaccines (glycoconjugate vaccines) can be made by attaching the saccharide to a carrier protein. See Plotkin, S. A., and Orenstein, W. A., Vaccines, 4th ed., Saunders, pages 959-987 (2004), which is incorporated herein by reference. These glycoconjugates induce a T-cell dependent immune response with memory and effect also in small children, while the non-conjugated CPS generally fails to provide either a memory effect in adults or any substantial immunogenic effect in infants. The development of type A vaccines has been considered especially difficult, due to the inherent instability of the anomeric phosphate diester linkages that are part of the CPS. The repeating unit of type A is a monosaccharide, 2-acetamido-2-deoxy-α-D-mannopyranose linked 1→6 via a phosphodiester bridge (FIG. 1). In the native polysaccharide the 3-OH is acetylated to an extent of about 80%. The immunological importance of this acetylation has not been completely investigated, but there are indications that it is not of major significance.
Neisseria meningitidis serogroup A causes epidemic outbreaks of meningitis, mainly in parts of Africa south of the Sahara in the so-called meningitis belt. In the meningitis belt the estimated incidence for the period 1970-1992 was about 800,000 cases. See Plotkin, S. A., and Orenstein, W. A., Vaccines, 4th ed., Saunders, pages 959-987 (2004). The epidemic outbreaks of meningitis are devastating for the region, so an effective vaccine is urgently needed. The hope is of course, that the development of a good vaccine, in combination with efforts similar to the ones against smallpox performed by WHO in the 1960's and 70's could likewise eliminate meningitis caused by N. meningitides serotype A. Vaccinations are a much more cost effective way of controlling a disease than treatment with antibiotics and other therapies, and cost is especially important in the developing world.
Vaccines prepared from the polysaccharide coating on the bacterium, its capsular polysaccharide, are effective in adults. Exposure to this polysaccharide causes adults to develop an immunogenic response that protects against meningitis caused by N. meningitidis. A big limitation with such vaccines, though, is that the immune system of children under around two years of age does not respond to most polysaccharide antigens. Unfortunately, this is the age group at greatest risk for bacterial meningitis. Thus, the polysaccharide vaccines are of no use in young children. Furthermore, even in older children and adults, these vaccines induce only short-term immunity. Protection decreases rapidly and is generally gone by around two years after vaccination.
Polysaccharides like the N. meningitidis CPS are T-cell independent antigens, which means that they can give an immune response without the involvement of T-cells (thymus-derived cells). This response lacks several important properties that characterize the T-cell dependent immune response, such as immunological memory, class-switch from IgM to IgG, and affinity maturation. If the polysaccharide part is connected to a carrier protein, however, it triggers a cellular immune response that creates memory effect, and also gives protection in young children. Such polysaccharides linked to carrier proteins are often referred to as glycoconjugates, and are especially valuable as vaccines.
Glycoconjugate vaccines are so called because their production involves the conjugation of a polysaccharide antigen or other glycosidic antigen to a carrier protein. The saccharide moiety in glycoconjugate vaccines is usually a functionalized bacterial CPS, but it can also be synthetic. Synthetic carbohydrate structures have a number of potential advantages over those based on carbohydrates from natural sources. Naturally derived carbohydrates are heterogeneous mixtures and may include small amounts of natural impurities and contaminants. In contrast, synthetic carbohydrates can be produced as homogeneous single compounds in a controlled manner, with little or no batch-to-batch variability. Another advantage of synthetic structures is that they can be made to include functional groups for derivitization or modifications of the carbohydrate moiety that are difficult or impossible to perform on the native material. The carrier protein is an important factor in the modulation of the immunogenicity. Various carriers have been used for conjugation, and the best results have been achieved using detoxified versions of strongly immunogenic proteins like diphtheria and tetanus toxins, which have been approved for use in humans. See U.S. Pat. No. 4,354,170. It has also been shown that the immune system reacts more effectively when patients have already been immunized with the particular carrier protein.
A glycoconjugate vaccine is usually made by conjugating the native capsular polysaccharide structure of the bacterium to a suitable carrier protein. However there have been problems with that approach due to the properties of the polysaccharide that encapsulates N. meningitidis. Its phosphodiester linkage can degrade under the conditions necessary for attachment of the polysaccharide to proteins, and even after preparation, glycoconjugates of the native CPS tend to degrade during storage.
Phosphodiesters are normally quite stabile, but in the capsular polysaccharide of N. meningitidis, the phosphodiester is linked to the anomeric center of a carbohydrate residue. Thus one oxygen of the phosphodiester is also part of an acetal linkage, which makes it susceptible to hydrolytic cleavage catalyzed by electrophiles such as acid or metal ions. Cleavage of this bond breaks the polysaccharide down into smaller pieces. Unfortunately, during the manipulations required to form a glycoconjugate, or even in a vaccine formulation, the CPS of N. meningitidis A is subject to such degradation, rendering it difficult to make and store effective vaccines comprising this particular CPS.
One way of making the phosphodiester linkage more stable is to eliminate the oxygen between the phosphorus and the anomeric oxygen, so that portion of the linkage is no longer susceptible to cleavage by electrophilic hydrolysis. The exocyclic oxygen at the anomeric center can be replaced with an isosteric carbon atom, (CH2), transforming the phosphodiester into its C-phosphonate analogue. This should produce a stabilized version of the antigenic polysaccharide. One investigation directed toward this approach has recently been published. Torres-Sanchez, M. I., et al., Synlett (2005) 7:1147-1151. However, the authors did not assess the activity of their compounds or disclose an oligomer of more than two mannose units. Furthermore, their synthesis approach provided only the beta anomer at the position where the oligosaccharide is intended to link to a protein, while the native CPS of N. meningitidis only contains alpha-linkages. Thus there remains a need for alpha-linked glycoconjugates having stabilized linkages between the mannose units and for methods to synthesize them.
Another approach is to stabilize the phosphodiester linkages using inductive effects from nearby substituents to reduce the electron density at the anomeric oxygens; this, too, should slow the anticipated electrophilic degradation mechanisms. The electron-attracting property of the substituents can be maintained while an oligosaccharide is constructed, and perhaps also while it is conjugated to a protein, then removed once the molecule no longer needs to be exposed to destructive conditions.
The present invention includes each of these approaches as well as combinations thereof. One aspect of the invention thus provides an efficient synthesis of a C-phosphonate analogue of an oligomeric form of the repeating unit of the N. meningitidis type A capsular polysaccharide, needed for vaccine development. See Bundle, D. R., et al., J. Biol. Chem. (1974) 249:2275-2281. The invention particularly provides methods for introducing a spacer moiety through which the oligosaccharides of the invention can be conjugated to a protein to make a glycoconjugate vaccine, and it orients the spacer moiety in the alpha anomeric configuration. FIG. 2 illustrates a compound having the desired alpha configuration at this center and one having the beta configuration. Since the natural CPS of the target organism is a fully alpha-linked oligomer of mannose units, it is especially desirable to provide the same alpha-linked configuration in a synthetic immunogen. Even if all of the linkages between mannose units in a synthetic oligomer portion of a glycoconjugate are in the alpha configuration, the configuration at the center through which the oligosaccharide is linked to the protein can be particularly influential on the immunogenic effects when the oligosaccharide portion of the glycoconjugate contains fewer than about 10 mannose units.
The present invention thus provides such compounds and methods for the preparation of these compounds. The crucial coupling step is often performed using a C-phosphonate mono-ester and Mitsunobu conditions. While it may be impractical to prepare polysaccharides rivaling the natural CPS in size, that is not necessary: even oligosaccharides only a few mannose-units in length can elicit an immune response. And synthetic oligosaccharides offer the advantages of selective linkage mechanisms and increased stability in vivo, each of which should enhance their immunogenic effectiveness in vivo.
Another aspect of the invention provides methods to stabilize the mannose units so that a phosphodiester linkage, the linkage present in the native capsular polysaccharide, can be used. It includes compounds and methods that provide stabilization to facilitate preparation of the protein conjugates of the oligosaccharides. The stabilization is provided by an electron-attracting group, an azide, at C-2 of at least one of the mannose units in a polysaccharide, which increases the polysaccharide's stability so that it can be conjugated to a protein. After conjugation, the stabilization is less important, and at that point the azide is typically reduced to an amine and acylated, which provides the 2-acetylamino group that is part of the recurring mannose unit in the N. meningitidis A capsular polysaccharide.
Where appropriate, it is also possible to mix the two stabilization methods; thus an oligosaccharide of the invention may include azide-containing mannose units that are linked by a phosphodiester at the adjacent anomeric center in combination with phosphonate-linked mannose units. This combination may provide additional stabilization, depending on the orientation of the phosphonate; or it may provide synthetic advantages such as increased yields and it may avoid complications that the acetylamine substituent can cause.
In other aspects, the invention provides methods to synthesize key precursors for these compounds, pharmaceutical compositions containing them, and methods to use them to manufacture a medicament. The compounds, compositions and medicaments may be administered to a subject to induce an immunogenic response in the subject, which is typically a human.